Numerous products of commercial interest, such as intermediates of L-ascorbic acid, have been produced biocatalytically in genetically engineered host cells. L-Ascorbic acid (vitamin C, ASA) finds use in the pharmaceutical and food industry as a vitamin and antioxidant. The synthesis of ASA has received considerable attention over many years due to its relatively large market volume and high value as a specialty chemical.
The Reichstein-Grussner method, a chemical synthesis route from glucose to ASA, was first disclosed in 1934 (Helv. Chim. Acta 17:311–328). Lazarus et al. (1989, “Vitamin C: Bioconversion via a Recombinant DNA Approach”, Genetics and Molecular Biology of Industrial Microorganisms, American Society for Microbiology, Wash. D.C. Edited by C. L. Hershberger) disclose a bioconversion method for production of an intermediate of ASA, 2-keto-L-gulonic acid (2-KLG, KLG) which can be chemically converted to ASA. This bioconversion of carbon source to KLG involves a variety of intermediates, the enzymatic process being associated with co-factor dependent 2,5-DKG reductase activity (2,5-DKGR or DKGR).
Many bacterial species have been found to contain DKGR, particularly members of the Coryneform group, including the genera Corynebacterium, Brevibacterium, and Arthrobacter. DKGR obtained from Corynebacterium sp. strain SHS752001 is described in Grindley et al. (1988) Applied and Environmental Microbiology 54: 1770–1775. DKGR from Erwinia herbicola is disclosed in U.S. Pat. No. 5,008,193 to Anderson et al. Other reductases are disclosed in U.S. Pat. Nos. 5,795,761; 5,376,544; 5,583,025; 4,757,012; 4,758,514; 5,004,690; and 5,032,514.
Host cells having mutations in enzymes involved in glycolysis have been described. Yeast having mutations in glucokinase are described in Harrod, et al. (1997) J. Ind. Microbiol. Biotechnol. 18:379–383; Wedlock, et al. (1989) J. Gen. Microbiol. 135: 2013–2018; and Walsh et al. (1983) J. Bacteriol. 154:1002–1004. Bacteria deficient in glucokinase have been described. Pediococcus sp. deficient in glucokinase is described in Japanese patent publication JP 4267860. Bacillus sphaericus lacking glucokinase is described in Russell et al. (1989) Appl. Environ. Microbiol. 55: 294–297. Penicillium chrysogenum deficient in glucokinase is described in Barredo et al. (1988) Antimicrob. Agents-Chemother 32: 1061–1067. A glucokinase-deficient mutant of Zymomonas mobilis is described in DiMarco et al. (1985) Appl. Environ. Microbiol. 49:151–157.
Bacteria which ferment glucose through the Embden-Meyerhof pathway, such as members of Enterobacteriacea and Vibrionaceae, are described in Bouvet, et al. (1989) International Journal of Systematic Bacteriology, p. 61–67. Pathways for metabolism of ketoaldonic acids in Erwinia sp. are described in Truesdell, et al, (1991) Journal of Bacteriology, pp 6651–6656.
However, most of the current methodologies utilized to produce compounds over-express the products. There are still problems remaining concerning the diversion of substrates used to produce the desired end-product by the cell for metabolic (catabolic) purposes, reducing the efficiency and overall production of thereof. Thus, there remains a need for improved methods for the production of products through pathways which are coupled to the metabolic pathways of host cells. The present invention addresses that need.
All publications cited herein are hereby incorporated by reference in their entirety.